Cytoplasmic dynein heavy chain is an essential gene product in Dictyostelium.
We describe here three different approaches to perturb cytoplasmic dynein heavy chain (DHC) gene function in Dictyostelium: integration of a marker into the heavy chain coding sequence by homologous recombination to disrupt transcription, expression of antisense RNA to inhibit translation, and expression of a 158 kDa amino-terminal coding region to perturb the native protein organization. By homologous recombination, we fail to obtain cells that lack an intact DHC gene product. Cells containing antisense orientation plasmids (but not sense) appear to die 4 to 6 days following transformation. Plasmids designed to overexpress an amino-terminal region of the DHC result in substantially reduced transformation efficiency. When expressed at low levels, the truncated amino-terminal product appears capable of dimerizing with an intact heavy chain or with itself, essentially producing a cargo-binding domain lacking mechanochemical activity. This, in turn, likely competes with the native protein's function. These three approaches taken together indicate that the dynein heavy chain is an essential gene in Dictyostelium.
Koonce
M P
MP
Division of Molecular Medicine, Wadsworth Center, Albany, New York 12201-0509, USA. Michael.Koonce@wadsworth.org
Knecht
D A
DA
D000818:Animals; D003001:Cloning, Molecular; D004023:Dictyostelium / Q000201:enzymology* / Q000235:genetics* / Q000502:physiology; D004398:Dyneins / Q000096:biosynthesis / Q000235:genetics* / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D016372:RNA, Antisense / Q000096:biosynthesis; D011995:Recombination, Genetic
D016372:RNA, Antisense; D004398:Dyneins